The goal of this project is to define the molecular mechanisms involved in the replication of mammalian retroviruses and in particular, to understand factors which influence the early phase of virus replication. We have been using an in vitro approach to study events in reverse transcription and the role of the viral nucleocapsid protein (NC) in increasing the efficiency and specificity of viral DNA synthesis. We have shown that HIV-1 NC greatly stimulates the rate and extent of HIV-1 minus-strand transfer in vitro, in part by suppressing formation of self-priming products generated from minus-strand strong-stop DNA [(-) SSDNA]. NC exerts this effect by destabilizing the large TAR secondary structure at the 3' end of (-) SSDNA. The biological significance of this previously unrecognized NC function is supported by the finding that in endogenous reverse transcriptase (RT) assays with purified HIV-1 virions (which contain NC), self-priming products cannot be detected. In related work, we have found a drug which specifically targets minus-strand transfer in both in vitro and endogenous reactions. The potential importance of this drug for anti-HIV therapy is being investigated in experiments with HIV-1-infected cells. Other studies are focused on nucleic acid-protein interactions involved in initiation of DNA synthesis. Experiments with RT enzymes having alanine or aromatic substitutions in residues of the HIV-1 p66 "primer grip" region show that almost all of the mutants are defective in utilization of RNA plus- and minus-strand primers, but are able to extend DNA versions of these primers. We conclude that residues in the primer grip undergo unique interactions with the RNA primers, which may reflect specific recognition of the helical structure of a primer-template having RNA in the primer strand. In additional studies with the RNA polypurine tract plus-strand primer, we find that alanine-scanning mutations in the thumb subdomain affect RT positioning on the primer-template and lead to a loss of RNase H cleavage specificity. Our results also show that proper RT positioning does not depend on the distance from the ends of the primer or template.